Project Summary/Abstract: The primary objective of this application is, for the first time, to generate and characterize an innovative resource for ablating subsets of cells. This resource will facilitate studying the roles of different subtypes of cells and their interactions in pathogenesis, tissue repair, and regeneration in vivo. We propose to advance the resources of cell ablation by generating a novel tool for targeting the subsets of cell populations that are defined by dual phenotypical markers. Loss-of-function studies using conditional targeted cell ablation have been widely used to investigate cell function and interaction, tissue repair and differentiation in vivo though they target only one marker-labelled cell population. Advanced methodologies are changing and expediting the ways in which we identify and understand immune, neural, and other cell subpopulations. These cell subpopulations can be labelled by the multiple phenotypical markers. We also anticipate that a growing spectrum of immune or neural subpopulations will be discovered in The Era of the Brain Initiative and The Post-Human Genome Project Era. Although current tools are available for ablating a cell population, they cannot specifically target cell subpopulations because they can only target one maker, not dual-labelled cells. Therefore, there is a need to develop an instrument that is able to eliminate subsets of cells in animals for loss- of-function studies. To this end, we propose to generate an intermedilysin (ILY)-mediated cell subpopulation ablation model. ILY, a toxin secreted by Streptococcus intermedius (SI), exclusively binds to the human cell membrane protein CD59 (hCD59), but not to CD59 of any other species. Once bound, ILY rapidly and potently lyses the cells. We recently established a Cre-inducible hCD59 transgenic mouse line (ihCD59) where hCD59 expression only occurs after Cre-mediated recombination. Administration of ILY to various lines of Cre+/- ihCD59+/- mice resulted in rapid and specific ablation of immune, epithelial or neural cells without off-target effects. We also tested the ILY/ihCD59-mediated cell ablation method in several disease models to study immune cell functions, hepatocyte and/or biliary epithelial damage and regeneration, and neural cell damage. This line (ihCD59) targets only one marker-labelled cell population, and cannot be used for ablating subset cell population. To further advance this tool towards subset cell ablation, we propose to develop and characterize CAG (CMV early enhancer/chicken beta actin)-floxP-STOP-floxP-Frt-STOP-Frt (DihCD59) or a double inducible mouse strain where the transgene (hCD59) expression occurs only following Cre- and Flp-mediated combinational events. Specifically, we will examine our working hypotheses that 1) hCD59 expression will be specifically mediated in the targeted subpopulations by Cre- and Flp-mediated combinational events in DihCD59 or by generating triple (Cre+/Flp+/DihCD59+) transgene positive mice; and that 2) ILY injection to the triple transgene positive mice will ablate subpopulations of cells for further investigating their functions in vivo.